Human growth hormone detection antibody and preparation method thereof

Abstract

The invention discloses a human growth hormone detection antibody and a preparation method thereof. The amino acid sequence of the detection antibody is one of SEQ ID NO. 1 and SEQ ID NO. 2; the preparation method comprises the following steps: extracting RNA (Ribonucleic Acid), performing reverse transcription, amplifying obtained cDN A, inserting into escherichia coli, and constructing a human growth hormone primary library; the M13KO7 auxiliary bacteriophage invades the human growth hormone primary library to construct a bacteriophage primary library; carrying out three rounds of solid phase panning on the phage primary library, and carrying out monoclonal screening, ELISA detection and sequencing to obtain a target gene sequence; and synthesizing the target gene sequence into a prokaryotic expression plasmid, introducing the prokaryotic expression plasmid into escherichia coli, and adding IPTG for expression to obtain the detection antibody. The detection antibody has the characteristics of low economy, strong stability and simple subsequent operation, and solves the problem that animal immunization is always needed to prepare the antibody in the subsequent process in human growth hormone detection.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and more specifically, to a human growth hormone detection antibody and a preparation method thereof. Background Art

[0002] Growth hormone (Human Growth Hormone, hGH) is a peptide hormone secreted by the anterior lobe of the human pituitary gland, consisting of 191 amino acids; its main functions include promoting growth (stimulating the growth of bones and soft tissues, especially in children and adolescents), metabolic regulation (stimulating protein synthesis, fat breakdown, and sugar metabolism), and cell repair (participating in tissue repair and regeneration).

[0003] Growth hormone is highly used, but its detection is difficult and the operation is complex. Currently, the detection methods used include radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), liquid chromatography-mass spectrometry (LC-MS), and animal immunization method. Radioimmunoassay (RIA) uses radioactively labeled GH to compete with GH in the sample for binding to antibodies. This method uses radioactive substances, has safety hazards, complex operations, and long time consumption. Chemiluminescence immunoassay (CLIA) uses chemiluminescent substances to label antibodies and quantifies GH by the luminescence intensity. This method has high equipment costs, relies on imports, and high reagent costs. Liquid chromatography-mass spectrometry (LC-MS) separates GH by chromatography and detects its molecular weight and fragment ions by mass spectrometry. This method has expensive equipment, requires professional technical personnel to operate, and high costs. The animal immunization method injects growth hormone antigen into animals to obtain antibodies, and then obtains the corresponding detection antibodies through purification and labeling for ELISA experiments. This method has high animal costs, requires re-immunization of animals each time antibodies are obtained, has a long cycle for obtaining antibodies, and involves animal hazards. Therefore, it is necessary to develop a human growth hormone detection antibody with low economy, strong stability, simple subsequent operations, and the ability to solve the problem of always needing to prepare serum antibodies through animal immunization in detection. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a human growth hormone detection antibody and a preparation method thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A human growth hormone detection antibody, the amino acid sequence of the detection antibody is one of those shown in SEQ ID NO.1 and SEQ ID NO.2.

[0007] A preparation method of a human growth hormone detection antibody, comprising the following steps:

[0008] S1. Extract the RNA from the spleen and blood of Chiloscyllium plagiosum respectively and reverse transcribe it to obtain cDNA. According to the universal primers, amplify the cDNA to obtain the VNAR gene, and insert the VNAR gene into Escherichia coli to construct a primary library of human growth hormone;

[0009] S2. Use the M13KO7 helper phage to invade the primary library of human growth hormone, display the expressed antibody on the phage surface, and construct a primary phage library;

[0010] S3. Perform three rounds of solid-phase panning on the primary phage library, and obtain the target gene sequence VNAR through monoclonal screening, ELISA detection and sequencing operations;

[0011] S4. Synthesize the target gene sequence VNAR into a prokaryotic expression plasmid, introduce it into Escherichia coli and add IPTG for expression to obtain a detection antibody for human growth hormone.

[0012] The present invention is further configured that in step S1, the specific steps for extracting the RNA of the blood of Chiloscyllium plagiosum are as follows: Take the whole blood from the caudal vein of Chiloscyllium plagiosum, let it stand and then separate the lymphocytes. Add Trizol reagent to the lymphocytes, mix well and let it stand; add chloroform, shake and then incubate and centrifuge for stratification; Transfer the upper aqueous phase containing RNA to a new RNA-free EP tube, add isopropanol, mix well, let it stand and centrifuge; Add 75% ethanol to wash the precipitate after centrifugation; Centrifuge again, remove the supernatant, dry the RNA precipitate, and add RNA-free ddH 2 O for dissolution, and store in aliquots.

[0013] The present invention is further configured that in step S1, the specific steps for extracting the RNA of the spleen of Chiloscyllium plagiosum are as follows: Grind the spleen into a powder, take the spleen powder and add Trizol reagent, mix well and let it stand, then add chloroform, shake and then incubate and centrifuge for stratification; Transfer the upper aqueous phase containing RNA to a new RNA-free EP tube, add isopropanol, mix well, let it stand and centrifuge; Add 75% ethanol to wash the precipitate after centrifugation; Centrifuge again, remove the supernatant, dry the RNA precipitate, and add RNA-free ddH 2 O for dissolution, and store in aliquots.

[0014] The present invention is further configured such that step S2 specifically includes: inoculating the primary library bacterial solution without glycerol obtained in step S1 into 2×YT / Tet / Car liquid medium, inoculating two bottles in total, and shaking until the logarithmic phase; adding M13KO7 helper phage to each bottle and then performing shaking culture; adding Kan medium to each bottle and continuing shaking culture; then adding IPTG medium to each bottle, performing shaking culture overnight, followed by centrifugation and filtration; transferring the supernatant obtained by filtration to a new centrifuge tube, adding PEG8000-NaCl solution and mixing evenly, performing centrifugation after ice bath, discarding the supernatant, and resuspending the phage precipitate with PBS to obtain a phage solution, which is the primary phage library.

[0015] The present invention is further configured such that in step S3, the specific steps of the first round of panning are as follows: diluting human growth hormone to 5 μg / mL with PBS solution and coating an ELISA plate therewith; discarding the coating solution and washing the plate with 0.05% PBST solution; blocking the ELISA plate with 3% BSA blocking solution; pouring out the liquid in the plate and washing the plate with 0.05% PBST solution; taking a part of the phages obtained in step S2, diluting to a fixed volume with PBS solution, adding 3% BSA blocking solution to block the phages, then diluting to a fixed volume with PBS solution, adding the remaining phages obtained in step S2, slowly shaking and then pouring out the phage mixture liquid in the plate, and washing the plate with 0.1% PBST solution; adding elution solution for incubation to elute the bound phages; collecting the elution solution, diluting to a fixed volume with PBS solution, and adjusting the pH to 7.0 to obtain the phage library of the first round of panning;

[0016] The specific steps of the second round of panning are as follows: diluting human growth hormone to 2 μg / mL with PBS solution and using it as the coating solution to coat an ELISA plate; discarding the coating solution and washing the plate with 0.05% PBST solution; blocking the ELISA plate with 3% skim milk powder blocking solution; pouring out the liquid in the plate and washing the plate with 0.05% PBST solution; adding phages with a titer of 1 / 3 of the phages obtained in the first round of panning, diluting to a fixed volume with PBS solution, adding 3% skim milk powder blocking solution to block the phages, then diluting to a fixed volume with PBS solution, adding the remaining phages obtained in the first round of panning, slowly shaking and then pouring out the phage mixture liquid in the plate, and washing the plate with 0.25% PBST solution; adding elution solution for incubation to elute the bound phages; collecting the elution solution, diluting to a fixed volume with PBS solution, and adjusting the pH to 7.0 to obtain the phage library of the second round of panning;

[0017] The specific steps of the third round of panning are as follows: Dilute human growth hormone to 1 μg / mL with PBS solution and use it as the coating solution to coat the ELISA plate; discard the coating solution and wash the plate with 0.05% PBST solution; block the ELISA plate with 3% BSA blocking solution; pour out the liquid in the plate and wash the plate with 0.05% PBST solution; add phages with a titer of 1 / 3 of the phages obtained in the second round of panning, make up the volume with PBS solution, then add 3% BSA blocking solution to block the phages, make up the volume with PBS solution again, add the remaining phages obtained in the second round of panning, slowly shake and then pour out the phage mixture liquid in the plate, and wash the plate with 0.5% PBST solution; add the elution solution for incubation to elute the bound phages; collect the elution solution, make up the volume with PBS solution, and adjust the pH to 7.0 to obtain the phage library of the third round of panning.

[0018] The present invention is further configured such that in step S3, the specific steps of monoclonal screening are as follows: Pick 88 monoclonal colonies from the output plates of the three rounds of panning into a 96-well deep well plate, add 2×YT / Car medium to each well, cover with a sealing film, puncture holes in the film, and shake until the logarithmic phase; aspirate the bacterial liquid in each well of the deep well plate into a new 96-well deep well plate, then add 2×YT / Car medium to the new 96-well deep well plate, cover with a sealing film, label the new deep well plate, which is used as the split plate; add 2×YT / Car and IPTG to each well of the split deep well plate, shake overnight and then centrifuge, discard the supernatant, add TEST lysis solution to each well, vortex and mix evenly, lyse on ice, and then perform overnight culture.

[0019] The present invention is further configured such that step S4 is specifically as follows:

[0020] a. Using the amino acid sequence of the human growth hormone detection antibody shown in SEQ ID NO.1 or SEQ ID NO.2 as a template, construct a VNAR recombinant plasmid, transfect the synthetic plasmid into BL21 competent cells by heat shock method, select monoclonal colonies through an LB plate for culture and freezing to obtain an expression strain;

[0021] b. Inoculate the obtained expression strain into a fermenter containing LB liquid medium for fermentation culture. After culturing until OD600 = 40, adjust the culture temperature to 16°C, add IPTG for overnight induction, centrifuge the induced bacterial liquid to obtain bacterial cells, resuspend the bacterial cells in Tris solution, perform cell wall breaking and centrifugation treatment, and collect the supernatant for filtration;

[0022] c. Perform elution and purification on the supernatant obtained in step b to obtain the human growth hormone VNAR antibody.

[0023] The present invention is further configured such that in step b, the inoculation amount of the expression strain is 1 ml; add IPTG to a final concentration of 0.5 mM.

[0024] The present invention is further configured such that step c specifically includes: taking out the GSTrap HP chromatography column, rinsing it thoroughly with deionized water and then equilibrating it with the equilibration buffer; loading the filtered supernatant sample onto the chromatography column; after the loading is completed, washing the chromatography column with the binding buffer until the A280 absorbance returns to the baseline level; then eluting with the elution buffer, collecting the elution peak, observing the change in A280 absorbance, when an obvious elution peak appears, collecting the eluate to obtain the human growth hormone VNAR antibody;

[0025] Among them, both the equilibration buffer and the binding buffer are solutions containing 50 mM Tris-HCl and 150 mM NaCl, with pH = 7.4; the elution buffer is a solution containing 50 mM Tris-HCl, 150 mM NaCl and 10 mM reduced glutathione, with pH = 7.4.

[0026] In summary, the present invention has the following beneficial effects:

[0027] The human growth hormone detection antibody of the present invention has the characteristics of low cost, strong stability and simple subsequent operations. Moreover, by constructing a library of Escherichia coli capable of expressing the human growth hormone detection antibody and storing it at -80°C, when in use, taking out the Escherichia coli for cultivation and adding an inducer can obtain the human growth hormone detection antibody, which solves the problem that subsequent animal immunization for serum antibody preparation has been required in the detection of human growth hormone by the conventional animal immunization method. Description of the Drawings

[0028] Figure 1 It is the electrophoresis result diagram of the RNA of the spleen and blood of Chiloscyllium plagiosum in step 1 of Example 1;

[0029] Figure 2 It is the electrophoresis result diagram of the cDNA gene obtained in step 2 of Example 1;

[0030] Figure 3 It is the electrophoresis result diagram of the VNAR gene amplified from the vector in step 2 of Example 1;

[0031] Figure 4 It is the primary library diagram of human growth hormone in step 2 of Example 1;

[0032] Figure 5 It is the phage library diagram obtained from the first round of solid-phase panning in Example 1;

[0033] Figure 6 It is the phage library diagram obtained from the second round of solid-phase panning in Example 1;

[0034] Figure 7 It is the phage library diagram obtained from the third round of solid-phase panning in Example 1;

[0035] Figure 8It is the Elisa test result diagram in step 5 of Example 1;

[0036] Figure 9 It is the result diagram for detecting the effect of the antibody of the BL21-GST-VNAR1-HIS expression strain in Example 2. Detailed implementation manners

[0037] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] Example 1 Construction of phage display library

[0039] The main process for constructing the phage display library is: RNA extraction and reverse transcription → construction of the primary library → collection of the growth hormone primary library → construction of the phage library → three rounds of solid-phase panning of the phage library → monoclonal Elisa screening → obtaining human growth hormone VNAR.

[0040] 1. RNA extraction and reverse transcription

[0041] Blood was taken from the caudal vein of the bamboo shark and its immunized shark spleen was dissected. RNA was extracted and reverse transcribed respectively to obtain cDNA.

[0042] The specific steps for extracting RNA from the blood of Chiloscyllium plagiosum are as follows: Take 12 mL of whole blood from the caudal vein of Chiloscyllium plagiosum. After allowing the blood to stand at room temperature for 2 hours, isolate PBMC cells (lymphocytes). Add 1 mL of Trizol reagent (RNA extraction reagent) to the lymphocytes, and mix well by pipetting up and down. Let it stand at room temperature for 5 - 10 min; Add chloroform in a ratio of 200 μl of chloroform per 1 mL of Trizol reagent, cover the tube cap, place it in a shaker and shake vigorously for 15 s, then incubate at room temperature for 5 - 10 min; After incubation, centrifuge the sample at 4°C and 12,000×g for 15 min to separate the mixture into a red phenol-chloroform lower layer, an intermediate layer, and a colorless upper aqueous phase; Transfer the upper aqueous phase containing RNA to a new RNA-free EP tube. Add isopropanol in a ratio of 0.5 mL of isopropanol per 1 mL of the upper aqueous phase, mix well and let it stand on ice for 10 min; Then centrifuge at 4°C and 12,000×g for 10 min (after centrifugation, the RNA precipitate is on the side and bottom of the tube); Add 1 mL of 75% ethanol (prepared with DEPC water) to wash the precipitate, and invert the EP tube to wash off the isopropanol remaining on the tube wall; Then centrifuge at 12,000×g at 4°C for 5 min, use a small pipette tip to remove the supernatant, air-dry the RNA precipitate in a laminar flow hood for about 5 min, and add 20 - 50 μl of RNA-free ddH 2 O (double-distilled water) to dissolve the RNA precipitate, and store it in aliquots.

[0043] The specific steps for extracting RNA from the spleen of Chiloscyllium plagiosum are as follows: Grind the spleen into a powder. Take 100 mg of the spleen powder, add 1 mL of Trizol reagent, and mix well by pipetting up and down. Let it stand at room temperature for 5 - 10 min; Add 200 μl of chloroform, cover the tube cap, place it in a shaker and shake vigorously for 15 s, then incubate at room temperature for 5 - 10 min; After incubation, centrifuge the sample at 4°C and 12,000×g for 15 min; Transfer the upper layer containing RNA to a new RNA-free EP tube. Add isopropanol in a ratio of 0.5 mL of isopropanol per 1 mL of the upper layer, mix well and let it stand on ice for 10 min; Then centrifuge at 4°C and 12,000×g for 10 min; Add 1 mL of 75% ethanol (prepared with DEPC water) to wash the precipitate, and invert the EP tube to wash off the isopropanol remaining on the tube wall; Then centrifuge at 12,000×g at 4°C for 5 min, use a small pipette tip to remove the supernatant, air-dry the RNA precipitate in a laminar flow hood for about 5 min, and add 20 - 50 μl of RNA-free ddH 2 O (double-distilled water) to dissolve the RNA precipitate, and store it in aliquots.

[0044] The blood and spleen RNA extraction and reverse transcription experiments of Chiloscyllium plagiosum were carried out separately according to the above methods. Two groups were conducted for both blood and spleen RNA extraction and reverse transcription, denoted as spleen group 1#, spleen group 2#, blood group 1#, and blood group 2#. The electrophoresis results of the extracted RNA are as Figure 1 shown.

[0045] 2. Construction and collection of the primary library of human growth hormone

[0046] 2.1 Mix the cDNA obtained by reverse transcription of spleen RNA and PBMC RNA. According to the universal primers (IgNAR1 / 2 / 3 / 4), amplify the VNAR gene, insert the VNAR gene into Escherichia coli, and obtain the primary library after transformation using the electrotransformation method.

[0047] 2.2 Collection of the primary library of human growth hormone: Randomly select 50 for monoclonal sequencing in the primary library of human growth hormone, collect the sequencing results, and ensure that more than 70% of VNAR can be obtained.

[0048] Mix the cDNA obtained after reverse transcription of spleen group 1# in step 1 with the cDNA obtained after reverse transcription of blood group 1#, mix the cDNA obtained after reverse transcription of spleen group 2# in step 1 with the cDNA obtained after reverse transcription of blood group 2#, then mix the two groups of mixed cDNA, and then carry out the construction and collection of the primary library of human growth hormone according to the methods of 2.1 and 2.2. During the experiment, electrophoresis tests were carried out on the two groups of mixed cDNA respectively, and the results are shown in Figure 2 ( Figure 2 In the figure, human growth hormone 1# represents the electrophoresis result diagram of the mixture of cDNA obtained by reverse transcription of spleen group 1# and cDNA obtained by reverse transcription of blood group 1#, Figure 2 In the figure, human growth hormone 2# represents the electrophoresis result diagram of the mixture of cDNA obtained by reverse transcription of spleen group 2# and cDNA obtained by reverse transcription of blood group 2#); During the experiment, electrophoresis tests were also carried out on the amplified VNAR gene (four groups of test experiments were set), and the test results are shown in Figure 3 . The diagram of the primary library of human growth hormone finally obtained by this step of operation is as Figure 4 shown (in the figure, each culture medium is set as: coating volume 10 μL, library capacity 3.2x10 9 , and the dilution multiples of the 6 culture media from left to right and top to bottom in the figure are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 ).

[0049] 3. Construction of the primary library of phage

[0050] Construct as follows:

[0051] 3.1 Inoculate 2 mL of the primary library bacterial solution of human growth hormone into 200 mL of 2×YT / Tet / Car liquid medium, inoculating two bottles in total, and shake at 37°C and 220 rpm until the logarithmic phase.

[0052] 3.2 Add 200 μL of M13KO7 helper phage to each bottle, and culture in a shaker at 37°C and 80 rpm for 0.5 h.

[0053] 3.3 Then add 200 μL of Kan medium to each bottle, and culture in a shaker at 37°C and 220 rpm for 0.5 h.

[0054] 3.4 Then add 100 μL of IPTG medium to each bottle, and culture in a shaker at 30°C and 220 rpm overnight.

[0055] 3.5 Centrifuge the bacterial solution that has been centrifuged overnight at 8000 rpm and 4°C for 40 min.

[0056] 3.6 Filter the supernatant through a 0.22-μm filter membrane into a new centrifuge tube, add the PEG8000-NaCl solution according to 1 / 5 of the supernatant volume, mix well, and incubate on ice for 1 h.

[0057] 3.7 Centrifuge at 8000 rpm and 4°C for 30 min, discard the supernatant, and invert the centrifuge tube on absorbent paper to fully remove the residual liquid;

[0058] 3.8 Resuspend the phage precipitate with 1 mL of PBS to obtain a phage solution, which is the primary phage library.

[0059] 3.9 Detect the titer of the obtained primary phage library.

[0060] 4. Three rounds of solid-phase panning of the phage library:

[0061] 4.1 First round of panning:

[0062] 4.1.1 Dilute human growth hormone with 0.01 M PBS to 5 μg / mL to obtain a coating solution, inject the coating solution into a 96-well ELISA plate, 100 μL / well, and coat overnight at 4°C.

[0063] 4.1.2 Discard the coating solution in the ELISA plate, and wash 3 times with 200 μL of 0.05% PBST solution.

[0064] 4.1.3 Prepare a 3% BSA blocking solution, add 200 μL of the blocking solution to each well, and block at 37°C for 1 h.

[0065] 4.1.4 Pour out the liquid in the plate, and wash 5 times with 200 μL of 0.05% PBST.

[0066] 4.1.5 Take 100 μL of the phages collected in step 3 and make up the volume to 600 μL with 0.01 M PBS solution; add 600 μL of 3% BSA blocking solution to block the phages, block at room temperature for 1 h, and then make up the volume to 5 mL with 0.01 M PBS solution.

[0067] 4.1.6 Add the remaining phages collected in step 3 and shake slowly at 25 °C for 1 h.

[0068] 4.1.7 Pour out the phage mixture liquid in the plate, wash 6 times with 0.1% PBST solution, add 100 μL of eluent to each well (add 0.75 g of Glycine, 0.05 g of BSA, dissolve in 45 mL of ddH 2 O, add 670 μL of 6 M HCl solution, add ddH 2 O to make up the volume to 50 mL, at this time the pH value is 2.2, filter and sterilize, store at 4 °C to obtain the eluent), incubate at room temperature for 5 min to elute the bound phages.

[0069] 4.1.8 Collect the eluent and make up the volume to 5 mL with 0.01 M PBS solution, immediately adjust the pH to 7.0 with the neutralizing solution (dissolve 1.21 g of Tris in 45 mL of ddH 2 O, add 225 μL of 6 M HCl solution, add ddH 2 O to make up the volume to 50 mL, at this time the pH value is 9.1. Filter and sterilize, store at 4 °C to obtain the neutralizing solution) to obtain the phage library after the first round of panning.

[0070] 4.2 Second round of panning:

[0071] 4.2.1 Dilute human growth hormone to 2 μg / mL with 0.01 M PBS solution to obtain the coating solution, inject the coating solution into a 96-well enzyme-linked immunosorbent assay (ELISA) plate, 100 μL per well, and coat overnight at 4 °C.

[0072] 4.2.2 Discard the coating solution in the ELISA plate and wash 3 times with 200 μL of 0.05% PBST solution.

[0073] 4.2.3 Prepare 3% skim milk powder blocking solution, add 200 μL of blocking solution to each well, and block at 37 °C for 1 h.

[0074] 4.2.4 Pour out the liquid in the plate and wash 5 times with 200 μL of 0.05% PBST.

[0075] 4.2.5 Add phages with a titer of 1 / 3 of the phages obtained from the first round of panning, make up the volume to 600 μL with 0.01 M PBS solution; add 600 μL of 3% skim milk powder blocking solution to block the phages, block at room temperature for 1 h, and then make up the volume to 5 mL with 0.01 M PBS solution.

[0076] 4.2.6 Add the remaining phages from the first round of panning, and gently shake at 25 °C for 45 min.

[0077] 4.2.7 Discard the phage mixture in the plate, wash 8 times with 0.25% PBST solution, add 100 μL of eluent to each well, and incubate at room temperature for 5 min to elute the bound phages.

[0078] 4.2.8 Collect the eluent and make up the volume to 5 mL, immediately adjust the pH to 7.0 with neutralizing solution to obtain the phage library for the second round of panning.

[0079] 4.3 Third round of panning:

[0080] 4.3.1 Dilute human growth hormone to 1 μg / mL with 0.01 M PBS solution to obtain the coating solution, inject the coating solution into a 96-well ELISA plate, 100 μL per well, and coat overnight at 4 °C.

[0081] 4.3.2 Discard the coating solution in the ELISA plate, and wash 3 times with 200 μL of 0.05% PBST solution.

[0082] 4.3.3 Add 200 μL of 3% BSA blocking solution to each well and block at 37 °C for 1 h.

[0083] 4.3.4 Discard the liquid in the plate and wash 5 times with 200 μL of 0.05% PBST.

[0084] 4.3.5 Add phages with a titer of 1 / 3 of the phages obtained in the second round, make up the volume to 600 μL with 0.01 M PBS solution; add 600 μL of 3% BSA blocking solution to block the phages, block at room temperature for 1 h, and then make up the volume to 5 mL with 0.01 M PBS solution.

[0085] 4.3.6 Add the remaining phages from the second round of panning, and gently shake at 25 °C for 30 min.

[0086] 4.3.7 Discard the phage mixture in the plate, wash 8 times with 0.5% PBST solution, add 100 μL of eluent to each well, and incubate at room temperature for 5 min to elute the bound phages.

[0087] 4.3.8 Collect the eluent and make up the volume to 5 mL, immediately adjust the pH to 7.0 with neutralizing solution to obtain the phage library for the third round of panning.

[0088] 4.4 Inoculate the MC1061F' bacterial solution (E. coli) into 30 mL of 2×YT / Tet liquid medium, and culture at 37 °C and 220 rpm until the logarithmic phase.

[0089] 4.5 Add the phages obtained from three rounds of panning to 30 mL of the bacterial liquid cultured to the logarithmic phase, culture at 37 °C and 150 rpm for 30 min, and conduct infection.

[0090] 4.6 Take 100 μL of the infected bacterial liquid and dilute it by 10-fold gradient, then coat it on a 2×YT / Tet / Car / 2% glucose plate for subsequent picking of monoclonal colonies.

[0091] The phages obtained from three rounds of solid-phase panning of the primary phage library obtained in Step 3 are shown respectively in Figures 5 - 7 .

[0092] 5. Monoclonal Screening, ELISA Detection, and Sequencing

[0093] 5.1 After three rounds of panning, pick 88 monoclonal colonies from the output plates of the third round of panning into a 96-deep well plate (do not use the 12th column), add 600 μL of 2×YT / Car medium to each well, cover with a sealing film, make holes in the film, write labels on the deep well plate, and shake at 37 °C and 220 rpm until the logarithmic phase;

[0094] 5.2 Pipette 100 μL of the bacterial liquid in each well of the deep well plate into a new 96-deep well plate, and then add 100 μL of 2×YT / Car medium to the new 96-deep well plate, cover with a sealing film, write labels on the new deep well plate, and this plate is the split plate.

[0095] 5.3 Add 100 μL of 2×YT / Car and 0.6 μL of IPTG to each well of the split plate deep well plate, and shake overnight at 30 °C and 220 rpm;

[0096] 5.4 The next day, centrifuge the split plate deep well plate at 4000 rpm for 20 min using a plate centrifuge, pour off the supernatant, add 300 μL of TEST lysis buffer to each well, vortex to mix evenly, lyse on ice for 1 h, shake once every 15 min during lysis, and then culture overnight at 4 °C;

[0097] 5.5 Coat the ELISA plate, mark the sample names on the ELISA plate (in the ELISA plate, number them in sequence as A1 - A12, B1 - B12, C1 - C12, D1 - D12, E1 - E12, F1 - F12, G1 - G12, H1 - H12, where A12, B12, C12, D12, E12, F12, G12, H12 are control wells, and the rest are induced clone wells);

[0098] 5.6 Conduct ELISA detection according to the following process (the test results are shown in Figure 8 );

[0099]

[0100] 5.7 Pipette the target cloned bacterial liquid (i.e., the cloned bacterial liquid corresponding to 1-13 in Figure 8 ) from the "bacterial plates" with higher ELISA readings into an EP tube. Label the EP tube lid and send it to Hangzhou Youkang Sequencing Company for sequencing. The sequencing primer is "p3Rd v-SeqF".

[0101] The amino acid sequences of some VNAR genes obtained by sequencing are shown in SEQ ID NO.1 - SEQ ID NO.7:

[0102] SEQ ID NO.1 ( Figure 8 the sequence obtained from the cloned bacterial liquid corresponding to 12 in

[0103] VEQTPTTTTKEAGESLTINCVLKGSSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAGYWIGYCSGIVGIDAAYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQI

[0104] SEQ ID NO.2 ( Figure 8 the sequence obtained from the cloned bacterial liquid corresponding to 4 in

[0105] VEQTPTTTTKEAGESLTINCVLKGSSYALCDTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAWPLVGGYCSVGYLYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0106] SEQ ID NO.3 ( Figure 8 the sequence obtained from the cloned bacterial liquid corresponding to 13 in

[0107] VEQTPTTTTKEAGESLTINCVLKGSSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAGYWIGYCSGIVGIDAAYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0108] SEQ ID NO.4 ( Figure 8 the sequence obtained from the cloned bacterial liquid corresponding to 2 in

[0109] VEQTPTTTTKEAGESLTINCVLKGNSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAGVWIGYCSGIVGIDAAYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0110] SEQ ID NO.5( Figure 8 The sequence measured from the cloned bacterial solution corresponding to 8 in

[0111] VEQTPTTTTKEAGESLTINCVLKGSSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAGGWIGYCSDIIGIDAAYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0112] SEQ ID NO.6( Figure 8 The sequence measured from the cloned bacterial solution corresponding to 11 in

[0113] VEQTPTTTTKEAGESLTINCVLKGSSYALCDTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYPALYCSRWDSARDYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0114] SEQ ID NO.7( Figure 8 The sequence measured from the cloned bacterial solution corresponding to 6 in

[0115] VEQTPTTTTKEAGESLTINCVLKGSNYALCDTYWYFTKKGATKKENLSNGGRYAETVNKASKSFSLRISDLRIEDSGTYHCKGSYNWERLLAHTYYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0116] Example 2 Construction, Fermentation Purification of VNAR Recombinant Plasmid and Detection of the Effect of the Expressed Strain Antibody

[0117] 1. Construction of VNAR Recombinant Plasmid

[0118] 1.1 Select two VNAR sequences (SEQ ID NO.1 and SEQ ID NO.2), named Sequence 1 and Sequence 2 respectively. Add a GST tag to the N-terminus and a 6xHIS tag to the C-terminus of the amino acid sequence of Sequence 1 to form the amino acid sequence shown in SEQ ID NO.8; add a GST tag to the N-terminus of the amino acid sequence of Sequence 2 to form the amino acid sequence shown in SEQ ID NO.9.

[0119] SEQ ID NO.8:

[0120] MKYLLPTAAAGLLLLAAQPAMAGGSGGSHHHHHHGGSGGGSMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKGGGVEQTPTTTTKEAGESLTINCVLKGSSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAGYWIGYCSGIVGIDAAYYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQI

[0121] SEQ ID NO.9:

[0122] MKYLLPTAAAGLLLLAAQPAMAGGSGGSMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKGGGVEQTPTTTTKEAGESLTINCVLKGSSYALCNTVWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAWPLVGGYCSVGYLYYEGGGTILTVKPGKQPSPPIISLHYSATEEQRANGFLQLI

[0123] 1.2 Convert these two sequences into DNA sequences and perform codon optimization. Integrate the two target genes into the pET22b(+) plasmid by double digestion with NdeⅠ and EcoRⅠ restriction enzyme sites respectively, and outsource the synthesis of plasmids pET22b(+)GST-VNAR1-HIS and pET22b(+)-GST-VNAR2.

[0124] 1.3 Transfect the two synthesized plasmids into BL21 competent cells by heat shock method respectively, select monoclonal cultures through LB plates (ampicillin resistance) and freeze them, and obtain BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 expression strains respectively.

[0125] 2. Fermentation

[0126] Ferment the BL21-GST-VNAR1-HIS expression strain and the BL21-GST-VNAR1 expression strain according to the following method respectively to obtain two kinds of supernatants:

[0127] 2.1 Take the preserved expression strains, pre-culture them in shake flasks and then inoculate them into a 5L fermenter containing LB liquid medium (control the inoculation amount of the expression strain to 1 ml). After culturing until OD600 = 40, adjust the culture temperature to 16°C, and add IPTG with a final concentration of 0.5 mM for overnight induction for 20 h.

[0128] 2.2 Centrifuge the induced bacterial solutions at 8000g for 10 min respectively to obtain the bacterial cells.

[0129] 2.3 Resuspend the bacterial cells at a mass ratio of wet cell weight to 20 mM Tris pH = 8.0 solution of 1:10, and perform cell wall breaking treatment on the bacterial cells twice under the condition of 1000 bar. After cell wall breaking, centrifuge the solution at 8000 g for 10 min, collect the supernatant and filter it through a 0.45 μm capsule filter.

[0130] 3. Purification of the supernatant after cell wall breaking

[0131] Purify the two supernatants obtained from the above fermentation steps respectively according to the following methods to obtain BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 expression strain antibodies:

[0132] 3.1 Solution preparation:

[0133] Binding buffer: 50 mM Tris-HCl, 150 mM NaCl, pH = 7.4.

[0134] Elution buffer: 50 mM Tris-HCl, 150 mM NaCl, 10 mM reduced glutathione, pH = 7.4.

[0135] Equilibration buffer: same as the binding buffer.

[0136] 3.2 Chromatographic column preparation

[0137] A. Take out the GSTrap HP column, rinse it with deionized water at a flow rate of 5 ml / min for 10 min, and rinse off the ethanol.

[0138] B. Equilibrate the chromatographic column with the equilibration buffer at 5 ml / min until the baseline is stable.

[0139] 3.3 Load the supernatant sample obtained by purification in step 2 onto the chromatographic column, with a loading volume of 500 ml and a loading flow rate set at 5 ml / min.

[0140] 3.4 After loading, wash the chromatographic column with the binding buffer at 5 ml / min until the A280 absorbance returns to the baseline level.

[0141] 3.5 Elute with the elution buffer at 2 ml / min, collect the elution peak, observe the change in A280 absorbance, and collect the eluate when an obvious elution peak appears.

[0142] 3.6 After elution, rinse the chromatographic column with the binding buffer at 5 ml / min for 10 min, then rinse it with 20% ethanol at 5 ml / min for 10 min, and store the chromatographic column in 20% ethanol.

[0143] 3.7 Replace the two eluents with an ultrafiltration tube into a solution of 20 mM PB pH 7.5, measure the protein concentration using the BCA method, and dilute it to 1 mg / ml with a solution of 20 mM PB pH 7.5.

[0144] 4. Detection of the effects of antibodies of BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 expression strains

[0145] Detect the effects of antibodies of BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 expression strains respectively according to the following method:

[0146] 4.1 Dilute the antibody to 50 μg / ml with the coating solution, add 100 μl / well of the diluted solution to the enzyme-linked immunosorbent assay (ELISA) plate and incubate overnight. Wash the ELISA plate the next day and block for 1-2 hours;

[0147] 4.2 Add human growth hormone to each well at 20 ng, 10 ng, 5 ng, 2.5 ng, 1.25 ng, 0.625 ng, and 0 ng in 100 μl aliquots with the dilution buffer, and incubate at 37 °C for 1 hour;

[0148] 4.3 Take out the ELISA plate, wash it with the washing solution, add 100 μl of the antibody diluted 1:1000 to each well, and incubate for 1 hour;

[0149] 4.4 Add HRP-labeled mouse anti-HIS monoclonal antibody (purchased from: Sangon Biotech) and develop color with TMB, terminate the reaction, and read the absorbance.

[0150] The results of the detection of the effects of antibodies of BL21-GST-VNAR1-HIS expression strain are shown in Table 1.

[0151] Table 1

[0152]

[0153] Example 3 Library construction and preservation of BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 expression strains

[0154] 3.1 Prepare 40% glycerol: Draw 12 ml of glycerol with a syringe, stir it well and dissolve it in 18 ml of distilled water for injection, and sterilize it by autoclaving (121 °C, 40 minutes) for later use.

[0155] 3.2 Mix BL21-GST-VNAR1-HIS and BL21-GST-VNAR1 expression strains with 40% glycerol at a volume ratio of 1:1 respectively, aliquot 1.0 ml / tube into cryotubes, and store them in an environment of -80 °C or below.

[0156] The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the concept of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A human growth hormone detection antibody, characterized in that: The amino acid sequence of the detection antibody is one of SEQ ID NO.1 and SEQ ID NO.

2.

2. A method for preparing a human growth hormone detection antibody as claimed in claim 1, characterized in that: The following steps are involved: S1. RNA from the spleen and blood of the striped bamboo shark was extracted and reverse transcribed to obtain cDNA. The cDNA was amplified using universal primers to obtain the VNAR gene, and the VNAR gene was inserted into Escherichia coli to construct a primary library of human growth hormone; S2, using M13KO7 to assist phage in invading the primary library of human growth hormone, displaying the expressed antibodies on the surface of phage, and constructing the primary phage library; S3, three rounds of solid phase panning were performed on the primary phage library, and the target gene sequence VNAR was obtained through monoclonal screening, ELISA detection and sequencing; S4. The target gene sequence VNAR is synthesized into a prokaryotic expression plasmid, introduced into Escherichia coli and IPTG is added for expression to obtain human growth hormone detection antibodies.

3. The method for preparing a human growth hormone detection antibody according to claim 2, characterized in that: In step S1, the specific steps of extracting RNA from the blood of the striped bamboo shark are as follows: taking whole blood from the tail vein of the striped bamboo shark, separating lymphocytes after standing, adding Trizol reagent to the lymphocytes, mixing, and standing; adding chloroform, shaking, incubating, and centrifuging to separate; transferring the upper aqueous phase containing RNA to a new RNA-free EP tube, adding isopropanol, mixing, standing, and centrifuging; adding 75% ethanol to wash the precipitate after centrifugation; Centrifuge again, remove the supernatant, dry the RNA precipitate, add RNA-free ddH2O to dissolve it, and store it in aliquots.

4. The method for preparing a human growth hormone detection antibody according to claim 2, characterized in that: In step S1, the specific steps of extracting RNA from the spleen of the striped bamboo shark are as follows: grind the spleen into powder, take the spleen powder and add Trizol reagent, mix and stand, then add chloroform, shake and incubate, centrifuge and separate; transfer the upper aqueous phase containing RNA to a new RNA-free EP tube, add isopropanol, mix and stand, centrifuge; add 75% ethanol to wash the precipitate after centrifugation; Centrifuge again, remove the supernatant, dry the RNA precipitate, add RNA-free ddH2O to dissolve it, and store it in aliquots.

5. The method for preparing a human growth hormone detection antibody according to claim 2, characterized in that: Step S2 is specifically as follows: the primary library bacterial liquid obtained in step S1 without the addition of glycerol is inoculated into 2×YT / Tet / Car liquid culture medium, two bottles are inoculated, and shaken to the logarithmic phase; M13KO7 helper phage is added to each bottle and then shaken for culture; Kan culture medium is added to each bottle and the shaking culture is continued; IPTG culture medium is then added to each bottle, and the shaken culture is performed overnight, followed by centrifugation and filtration; the supernatant obtained by filtration is transferred to a new centrifuge tube, PEG8000-NaCl solution is added to mix, ice bath is performed, centrifuged, the supernatant is discarded, and the phage precipitate is resuspended with PBS to obtain a phage solution, which is the primary phage library.

6. The method for preparing a human growth hormone detection antibody according to claim 2, characterized in that: In step S3, the specific steps of the first round of panning are: dilute human growth hormone to 5 μg / mL with PBS solution, and use it to coat the ELISA plate; discard the coating solution and wash the plate with 0.05% PBST solution; use 3% BSA blocking solution to block the ELISA plate; pour out the liquid in the plate and wash the plate with 0.05% PBST solution; take part of the phage obtained in step S2, add 3% BSA blocking solution to block the phage after making up the volume with PBS solution, and then make up the volume with PBS solution, add the remaining phage obtained in step S2, slowly shake and pour out the phage mixed liquid in the plate, and wash the plate with 0.1% PBST solution; add elution solution for incubation to elute the bound phage; collect the elution solution and make up the volume with PBS solution, adjust the pH to 7.0, and obtain the phage library of the first round of panning; The specific steps of the second round of selection are as follows: dilute human growth hormone to 2 μg / mL with PBS solution, and use this as the coating solution to coat the ELISA plate; The coating solution was discarded and the plate was washed with 0.05% PBST solution; the ELISA plate was blocked with 3% skimmed milk powder blocking solution; the liquid in the plate was poured out and the plate was washed with 0.05% PBST solution; phages with a titer of 1 / 3 of the phages obtained in the first round of panning were added, and the volume was fixed with PBS solution, and then 3% skimmed milk powder blocking solution was added to block the phages, and then the volume was fixed with PBS solution, and the remaining phages obtained in the first round of panning were added, and the phage mixed liquid in the plate was poured out after slow shaking, and the plate was washed with 0.25% PBST solution; the elution solution was added for incubation to elute the bound phages; the elution solution was collected and fixed with PBS solution, and the pH was adjusted to 7.0 to obtain the phage library for the second round of panning; The specific steps of the third round of selection are as follows: dilute human growth hormone to 1 μg / mL with PBS solution, and use this as the coating solution to coat the ELISA plate; The coating solution was discarded and the plate was washed with 0.05% PBST solution; the ELISA plate was blocked with 3% BSA blocking solution; the liquid in the plate was poured out and the plate was washed with 0.05% PBST solution; phages with a titer of 1 / 3 of the phages obtained in the second round of panning were added, the volume was fixed with PBS solution, and then 3% BSA blocking solution was added to block the phages, and the volume was fixed with PBS solution, and the remaining phages obtained in the second round of panning were added, and the phage mixed liquid in the plate was poured out after slow shaking, and the plate was washed with 0.5% PBST solution; the elution solution was added for incubation to elute the bound phages; the elution solution was collected and fixed to volume with PBS solution, and the pH was adjusted to 7.0 to obtain the phage library of the third round of panning.

7. The method for preparing a human growth hormone detection antibody according to claim 2, characterized in that: In step S3, the specific steps of monoclonal screening are as follows: 88 monoclones are selected from the output plates of the three rounds of panning and placed in a 96-deep-well plate, 2×YT / Car culture medium is added to each well, the sealing film is covered, holes are punched in the film, and the plate is shaken to the logarithmic phase; the bacterial solution in each well of the deep-well plate is aspirated to a new 96-deep-well plate, and 2×YT / Car culture medium is added to the new 96-deep-well plate, the sealing film is covered, and the new deep-well plate is labeled as a split plate; 2×YT / Car and IPTG are added to each well of the split-plate deep-well plate, the plate is shaken overnight and then centrifuged, the supernatant is discarded, TEST lysis solution is added to each well, the plate is vortexed and mixed, and then the plate is lysed on ice, and then cultured overnight.

8. The method for preparing a human growth hormone detection antibody according to claim 2, characterized in that: Step S4 is specifically as follows: a. Using the amino acid sequence of the human growth hormone detection antibody shown in SEQ ID NO.1 or SEQ ID NO.2 as a template, construct a VNAR recombinant plasmid, transfect the synthetic plasmid into BL21 competent cells by heat shock method, select single clones by LB plate, culture and freeze, and obtain an expression strain; b. The obtained expression strain was inoculated into a fermenter containing LB liquid medium for fermentation culture. After culturing to OD600 = 40, the culture temperature was adjusted to 16 ° C, IPTG was added for overnight induction, and the induced bacterial liquid was centrifuged to obtain bacterial cells, and the bacterial cells were resuspended in Tris solution, broken, centrifuged, and the supernatant was collected and filtered; c. Eluting and purifying the supernatant obtained in step b to obtain human growth hormone VNAR antibody.

9. The method for preparing a human growth hormone detection antibody according to claim 8, characterized in that: In step b, the inoculum volume of the expression strain is 1 ml; IPTG is added to a final concentration of 0.5 mM.

10. The method for preparing a human growth hormone detection antibody according to claim 8, characterized in that: Step c is specifically as follows: taking out the GSTrap HP chromatographic column, rinsing it with deionized water, and then balancing it with a balancing buffer; loading the filtered supernatant sample onto the chromatographic column; after loading, washing the chromatographic column with a binding buffer until the A280 absorbance returns to the baseline level; then eluting with an elution buffer, collecting the elution peak, observing the change in A280 absorbance, and collecting the eluate when an obvious elution peak appears, thereby obtaining the human growth hormone VNAR antibody; The equilibration buffer and the binding buffer are both solutions containing 50 mM Tris-HCl and 150 mM NaCl, pH = 7.4; the elution buffer is a solution containing 50 mM Tris-HCl, 150 mM NaCl and 10 mM reduced glutathione, pH = 7.4.

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